Method of assaying for agonists or antagonist of Dynorphin A binding to the MAS receptor

ABSTRACT

The present invention is directed to assays that can be used to screen for compounds that act as agonists or antagonists or inverse agonists of Dynorphin A and its analogues. The assays are based upon the binding of Dynorphin A and its analogues to the rat and human MAS receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/SE01/02854, filed Dec. 19, 2001, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application 60/257,977, filed Dec.22, 2000.

FIELD OF THE INVENTION

The present invention is directed to assay methods that can be used todetermine whether a test compound can be used to modulate the bindingand activity of Dynorphin A and analogues at the human MAS receptor.Compounds identified as being effective modulators have potential use astherapeutic agents in treating pain, neuropathic and inflammatorydisorders, learning and memory, anxiety disorders as well as theregulation of cardiovascular functions.

BACKGROUND OF THE INVENTION

A. Dynorphin A

Dynorphin was discovered in 1979 as being an endogenously potent opioidpeptide (PNAS, USA 76:6666–6670: 1979). The pharmacological actions ofdynorphins are quite vast. Dynorphin A and related peptides have beenshown to be moderately effective in non-thermal and mechanical analgesiawhen administered ICV (Dubner, R., Trends Neurosci. 15:96–103, 1992) andhave also been used in the clinic for the treatment of intractable painin cancer patients (Wen, H. L., Central and peripheral endorphins: basicand clinical aspects, New York: Raven Press; 1993:319–323). Dynorphinsare also known to have an effect on the cardiovascular system via thecentral and peripheral nervous systems (Dumont, M. 37:1–33, 1996).Moreover, Dynorphin A has an immunomodulatory activity. Whenadministered to mice, Dynorphin A enhanced phagocytosis in the mouseperitoneal macrophages. This phagocytic activity was not inhibited bynaloxone treatment suggesting the involvement of a non-opioid receptor(J. Neuroimmunol. 60:37–43, 1995).

Even though, Dynorphin A was first described as a potent opioid peptidewith selectivity for kappa opioid receptor, some of its pathological andphysiological actions have been proposed to be mediated by non-opioidreceptors. Hence, here we described for the first time that Dynorphin Ais able to activate and bind to the MAS receptor.

The human MAS oncogene receptor was first isolated in 1986 via atumorgenicity assay in nude mice (Young, M., Cell, 45:711–719, 1986).This receptor codes for a seven-transmembrane domain protein and withinits coding sequence a hallmark feature is present such as the NPY motifin the seventh transmembrane domain. The MAS receptor is expressed inthe ventral nervous system (CNS) with highest signal of the mRNAobserved in the hippocampus, cortex, cerebellum, piriform cortex andolfactory bulb. The mRNA for this receptor has also been detected inperipheral tissues including the testis, kidney, and heart (FEBSLetters, 357, 27–32, 1995). The expression of MAS receptor is highlyregulated during development and neuronal activity. Interestingly, micelacking the MAS protooncogene not only displayed an increased anxietybut long-term potentiation was prolonged without affecting the grossmorphology of the hippocampus (JBC, 273,11867–11873, 1998).

Hence, drugs targeted at the MAS receptor as agonists, antagonists orinverse agonists could be potentially used for treating problems oflong-term memory neuropathic and inflammatory disorders, as well ascardiovascular dysfunction.

B. G Protein-Coupled Receptors

G protein coupled receptors (GPCRs) constitute a family of proteinssharing a common structural organization characterized by anextracellular N-terminal end, seven hydrophobic alpha helices putativelyconstituting transmembrane domains and an intracellular C-terminaldomain. GPCRs bind a wide variety of ligands that trigger intracellularsignals through the activation of transducing G proteins (Caron, et al.,Rec. Prog. Horm. Res. 48:277–290 (1993); Freedman, et al., Rec. Prog.Horm. Res. 51:319–353 (1996)).

More than 300 GPCRs have been cloned thus far and it is generallyassumed that there exist well over 1,000 such receptors. Roughly 50–60%of all clinically relevant drugs act by modulating the functions ofvarious GPCRs (Gudermann, et al., J. Mol. Med. 73:51–63 (1995)). Many ofthe clinically relevant receptors are located in the central nervoussystem.

Among the GPCRs that have been identified and cloned is a gene thatencodes a protein homologous to the receptors of the DRR/RTA family. Wecalled this receptor MAS receptor and described the structure of thegene as it exists in humans. However, the endogenous ligand for thisfamily of receptors has not previously been identified (Cell: 45,711–719 1986, JBC 273,11867–11873 1998, WO 99/32519).

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that Dynorphin A andits analogues (Dynorphin A-amide and Dynorphin 1-13) activates the ratand human MAS receptor. Recombinant cells expressing either rat or humanMAS receptor can be used in conjunction with Dynorphin A and itsanalogues in screening assays designed to identify agonists inverseagonists and antagonists. Thus, in its first aspect, the invention isdirected to a method of assaying a test compound for its ability to bindto the MAS receptor. This is accomplished by incubating cells ormembranes expressing the receptor gene with Dynorphin A and itsanalogues and test compound. The extent to which the binding ofDynorphin A and its analogues is displaced is then determined.Radioligand assays or enzyme-linked immunosorbent assays may beperformed in which either Dynorphin A and its analogues or the testcompound is detectably labeled. Although any cell expressing MASreceptor may be used, a recombinant cell expressing a heterologous MASreceptor gene from either the rat or human is preferred. The term“heterologous” as used herein refers to any MAS receptor genetransfected into a cell, i.e., the term refers to any non-endogenous MASreceptor.

The invention also encompasses methods of determining if a test compoundis an agonist, antagonist, or inverse agonist of Dynorphin A and itsanalogues binding based upon a functional assay. One way to carry outsuch assays is to incubate a cell expressing MAS receptor with the testcompound and to then determine whether intracellular phospholipase C,adenyl cyclase activity or intracellular calcium concentrations aremodulated. Results should typically be compared with those obtained whenincubations are performed in a similar manner but in the absence of testcompound. In general, functional assays of this type will be performedin conjunction with binding assays of the sort described above. Thepreferred cell for use in the assays is a recombinant cell that has beentransformed with a MAS receptor gene. Test compounds that act asagonists should produce an increase in phospholipase C, decrease orincrease in adenylyl cyclase activity or increase in intracellularlevels of calcium. Inverse agonists may reduce phospholipase C activityor intracellular calcium levels, particularly if assays are performed inthe presence of a fixed amount of Dynorphin A and its analogues.Antagonists, should block the binding of Dynorphin A and its analoguesto the receptor but not produce the opposite response in terms ofphospholipase C activity or intracellular calcium that is the hallmarkof an inverse agonist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to assays that can be used to screencompounds for their ability to modulate the binding of Dynorphin A andits analogues to the rat and human MAS receptors. Any form of DynorphinA and its analogues or fragments may be used (Dynorphin A:YGGFLRRIRPKLKWDNQ-COOH or NH2(SEQ ID NO:1)). Those peptides may beobtained commercially (e.g Bachem, American Peptide Company) or can besynthesized using standard methodologies well known in the art. Thepeptide may be detectably labeled with radioisotopes such as ¹²⁵I or,alternatively, fluorescent or chemiluminescent labels can beincorporated. Also, the peptide can be joined to enzymes that arereadily detectable such as horseradish peroxidase.

The MAS receptor may be cloned from rat and/or human cells/tissues usingthe procedure described in Young, M., Cell, 45:711–719, 1986. TheExamples section provides a detailed description of a procedure that maybe used in cloning MAS receptor. Once obtained, the MAS receptorsequence should be incorporated into an expression vector with apromoter active in mammalian cells (Sambrook, et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989)). Examplesof promoters that may be used include that of the mouse metallothioneinI gene (Hamer, et al., J. Mol. Appl. Gen. 1:273–288 (1982)); theimmediate-early and TK promoter of herpes virus (Yao, et al., J. Virol.69:6249–6258 (1995); McKnight, Cell 31:355–365 (1982)); the SV 40 earlypromoter (Benoist, et al., Nature 290:304–310 (1981)); and, the CMVpromoter (Boshart, et al., Cell 41:521–530 (1985)). Vectors may alsoinclude enhancers and other regulatory elements.

Once expression vectors have been constructed, they can be introducedinto a mammalian cell line by methods such as calcium phosphateprecipitation, microinjection, electroporation, liposomal transfer,viral transfer or particle mediated gene transfer. Although othermammalian cells may be used, HEK-293 cells have been found to givesuccessful results and a procedure for expressing MAS receptor in thesecells is described in the Examples section. Standard procedures forselecting cells and for assaying them for the expression of MAS receptor(e.g., by Northern analysis) may be performed.

Once the Dynorphin A (or an analogue) peptide and cells expressing therat and human MAS receptors have been obtained, assays may be performedto determine whether test compounds have any effect on binding. A widevariety of different types of assays can be performed using standardmethods well known in the art. For example, in radioligand bindingassays, cells expressing MAS receptor are incubated with Dynorphin A andits analogues and with a compound being tested for binding activity. Thepreferred source of MAS receptor is recombinantly transformed HEK-293cells. Other cells may also be used provided they do not express otherproteins that strongly bind Dynorphin A and its analogues. This caneasily be determined by performing binding assays on cells transformedwith MAS receptor and comparing the results obtained with those obtainedusing their non-transformed counterparts.

Assays may be performed using either intact cells or with membranesprepared from the cells (see e.g., Wang, et al., Proc. Natl. Acad. Sci.U.S.A. 90:10230–10234 (1993)). As suggested above, the membranes, orcells, are incubated with Dynorphin A and its analogues and with apreparation of the compound being tested. After binding is complete,receptor is separated from the solution containing ligand and testcompound, e.g., by filtration, and the amount of binding that hasoccurred is determined. Preferably, the ligand used is detectablylabeled with a radioisotope such as ¹²⁵I. However, if desired, othertypes of labels can also be used. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin o-phthalaldehydeand fluorescamine. Useful chemiluminescent compounds include luminol,isoluminol, theromatic of acridinium ester, imidazole, acridinium salt,and oxalate ester.

Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabeled ligand. Forexample, labelled Dynorphin A and its analogues may be incubated withreceptor and test compound in the presence of a thousandfold excess ofunlabeled Dynorphin A and/or its analogues. Nonspecific binding shouldbe subtracted from total binding, i.e., binding in the absence ofunlabeled ligand, to arrive at the specific binding for each sampletested. Other steps such as washing, stirring, shaking, filtering andthe like may be included in the assays as necessary. Typically, washsteps are included after the separation of membrane-bound ligand fromligand remaining in solution and prior to quantitation of the amount ofligand bound, e.g., by counting radioactive isotope. The specificbinding obtained in the presence of test compound is compared with thatobtained in the presence of labeled ligand alone to determine the extentto which the test compound has displaced receptor binding.

In performing binding assays, care must be taken to avoid artifactswhich may make it appear that a test compound is interacting withreceptor when, in fact, binding is being inhibited by some othermechanism. For example, the compound being tested should be in a bufferwhich does not itself substantially inhibit the binding of Dynorphin Aand its analogues and should, preferably, be tested at several differentconcentrations. Preparations of test compound should also be examinedfor proteolytic activity and it is desirable that antiproteases beincluded in assays. Finally, it is highly desirable that compoundsidentified as displacing the binding of Dynorphin A and its analogues bereexamined in a concentration range sufficient to perform a Scatchardanalysis of the results. This type of analysis is well known in the artand can be used for determining the affinity of a test compound forreceptor (see e.g., Ausubel, et al., Current Protocols and MolecularBiology, 11.2.1–11.2.19 (1993)); Laboratory Techniques in Biochemistryand Molecular Biology, Work, et al., Ed. N.Y. (1978)). Computer programsmay be used to help in the analysis of results (e.g., Munson, P.,Methods Enzymol. 92:543–577 (1983)).

Depending upon their effect on the activity of the receptor, agents thatinhibit the binding of Dynorphin A and its analogues to receptor may beeither agonists or antagonists. Activation of receptor may be monitoredusing a number of different methods. For example, phospholipase C assaysmay be performed by growing cells in wells of a microtiter plate andthen incubating the wells in the presence or absence of test compoundtotal inositol phosphates (IP) may then be extracted in resin columns,and resuspended in assay buffer. Assay of IP thus recovered can becarried out using any method for determining IP concentration.Typically, phospholipase C assays will be performed separately frombinding assays, but it may also be possible to perform bindingphospholipase C assays on a single preparation of cells.

Activation of receptor may also be determined based upon a measurementof intracellular calcium concentration. For example, transformed HEK-293cells may be grown on glass cover slides to confluence. After rinsing,they may be incubated in the presence of an agent such as Fluo-3, Fluo-4and FURA-2 AM (Molecular Probe F-1221). After rinsing and furtherincubation, calcium displacement may be measured using a photometer.Other types of assays for determining intracellular calciumconcentrations are well known in the art and may also be employed.

Assays that measure the intrinsic activity of the receptor, such asthose based upon inositol phosphate measurement, may be used in order todetermine the activity of inverse agonists. Unlike antagonists whichblock the activity of agonists but produce no activity on their own,inverse agonists produce a biological response diametrically opposed tothe response produced by an agonist. For example, if an agonist promotedan increase in intracellular calcium, an inverse agonist would decreaseintracellular calcium levels.

The radioligand and cell activation assays discussed above provideexamples of the types of assays that can be used for determining whethera particular test compound alters the binding of Dynorphin A and itsanalogues to the human/rat MAS receptors and acts as an agonist orantagonist. There are many variations on these assays that arecompatible with the present invention. Such assays may involve the useof labelled antibodies as a means for detecting Dynorphin A and itsanalogues that has bound to receptor or may take the form of thefluorescent imaging plate reader assay (FLIPR) as described in theExamples section herein.

EXAMPLES

I. Methods

Preparation of Clone Rat and Human MAS Receptor:

See description in: Cell 1986 Jun. 6;45(5):711–9

Expression

HEK-293 cells were transfected with a mammalian expression constructcoding for the rat and human MAS receptor (pcDNA 3.1 vector, Invitrogen)using the Superfect reagent (Qiagen). A stable receptor pool of MASreceptor was developed by applying a selection marker (G418, 0.9 mg/ml)and the cells were maintained in this selection medium. The presence ofmRNA specific for clone MAS receptor was assessed by Northern blotanalysis and by the reverse transcriptase polymerase chain reaction(RT-PCR).

Ligands

In order to identify the ligand of clone rat and human MAS receptor, acollection of peptide and non-peptide ligands was obtained fromcommercial sources (Sigma, CalBiochem, American Peptide Company, Bachem,RBI, Phoenix). The compounds were dissolved in water/DMSO at 3 μM andplaced in 96 well microplates. A total of 1000 compounds (peptides andnon-peptides) were prepared and tested.

Assays

1) FLIPR Assay

A functional assay was performed with FLIPR (Fluorescent Imaging PlateReader, Molecular Devices) using the fluorescent calcium indicatorFluo-3 (Molecular Probes) on a 96 well platform. HEK-293 cells, eitherexpressing the receptor or wild type cells, were loaded with Fluo-3 asfollows. Stable HEK-293 clones expressing rat and human MAS receptor orparental cells were plated at a density of 10,000 cells/well in a 96well plate. On the day of the experiment, the MAS receptor cells wereloaded with fluorescent solution (Dulbecco's modified medium with 10%fetal bovine serum containing 4 μM Fluo-3 and 20% pluronic acid). Thecells were incubated at 37° C. for one hour in a humidified chamber.Following the incubation step, cells were washed five times in Hanks'with 20 mM Hepes and 0.1% BSA (pH 7.4). The cells were analyzed usingthe FLIPR system to measure the mobilization of intracellular calcium inresponse to different compounds.

2) Binding Assay

Membranes of HEK-293 cells, either expressing the receptor or wild typecells were prepared as previously described and frozen at −80° C. On theday of the experiment membranes were homogenized in a buffer containing50 mM TRIS/HCl ph 7.4, 5 mM MgCl2, EDTA 2 mM, PMSF 0.1 mM, BSA 1 mg/mlusing a B Dounce homogenizer and incubated 1 hour at room temperaturewith different concentrations of ¹²⁵I-Dynorphin A-NH2 and with(non-specific binding) or without (specific binding) 1 μM ofnon-labelled Dynorphin A-NH2. The ¹²⁵I-Dynorphin A-NH2 bound wascollected by filtration through Whatman GF/B filters presoaked in PEI.The filters were rinsed three times with 2.5 ml of the cold Tris/MgCl₂buffer and then counted using a TopCount NXT (Packard). Protein wasmeasured using a Bio-Rad dye reagent.

II. Results

1) FLIPR Results

HEK-293 cells endogenously express some GPCRs such as bradykinin andPACAP receptors which can be used as internal controls for assays. Thebackground signal was established with all of the compounds in theparental HEK-293 cells (non-transfected) using the FLIPR assay. HEK-293cells expressing the clone MAS receptor were stimulated with allcompounds and calcium responses were compared with those in parentalHEK-293 cells. Three compounds, (Dynorphin A, Dynorphin A-NH2 andDynorphin A 1-13), consistently elicited signals in the transformedcells but not in the wild type cells. This indicates that Dynorphin Aand its analogues are interacting with the recombinantly expressedreceptors. Confirmation of this conclusion was obtained by theobservation of a dose-response relationship with Dynorphin A and itsanalogues in the cells transfected with MAS receptor, but not in thenon-transfected cells or in cells transfected with other orphanreceptors. Thus, it has been established that clone rat and human MASreceptor are specific receptors Dynorphin A and its analogues. The ratand human MAS receptors can be used to screen compounds which eithermimic the action of Dynorphin A and its analogues (agonists) orantagonize the action of Dynorphin A and its analogues (antagonists).

Screening assays can be performed using the FLIPR assay described above.

2) Binding Results

HEK 293S membranes expressing the cloned MAS receptor showed specificbinding for 125I-Dynorphin A-NH2 No specific binding was observed inuntransfected HEK 293S membranes.

Screening assays can be performed using binding assay described above.Other assays that can be used include the GTPase assay, adenylyl cyclaseassays, assays measuring inositol phosphates, and reporter gene assays(e.g., those utilizing luciferase, aqueorin, alkaline phosphatase,etc.).

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by those ofskill in the art that the invention may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. A method for identifying a compound that is able to bind to the rator human MAS receptor, comprising: a) providing a cell or membrane froma cell transformed with a heterologous rat or human MAS receptor gene,said cell expressing the rat or human MAS receptor; b) incubating thecell or membrane with Dynorphin A or Dynorphin A-amide or Dynorphin A1-13 in the presence and absence of a test compound; and c) determiningbinding of Dynorphin A or Dynorphin A-amide or Dynorphin A 1-13 to thecell or membrane, wherein a decrease in binding in the presence of thecompound as compared with binding in the absence or the compoundindicates that the compound is able to bind to the rat or human MASreceptor.
 2. The method of claim 1, wherein the Dynorphin A or DynorphinA-amide or Dynorphin A 1-13 or the test compound is radioactivelylabeled.
 3. The method of claim 1, wherein the Dynorphin A or DynorphinA-amide or Dynorphin A 1-13 or the test compound is joined to an enzyme.4. A method for identifying a compound that increases or decreases theactivity of the rat or human MAS receptor, comprising: a) providing acell or membrane from a cell transformed with a heterologous rat orhuman MAS receptor gene, said cell expressing the rat or human MASreceptor; b) incubating the cell or membrane with Dynorphin A orDynorphin A-amide or Dynorphin A 1-13, and a test compound; c)determining intracellular concentration of calcium of said cell; and d)comparing the results obtained in step c) with the results obtained witha control in the absence or said test compound; wherein a change inintracellular concentration of calcium of said cell in the presence ofsaid compound as compared with intracellular concentration of calcium ofsaid cell in the absence of said compound indicates that said compoundincreases or decreases MAS receptor activity.